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Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria

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Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria Journal of Agricultural Science and Technology A 5 (2015) 30-39 doi: 10.17265/2161-6256/2015.01.005 D DAVID PUBLISHING Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria Nataliya Vladimirovna Punina1, Mikhail Anatolievich Remnev2 and Alexey Fedorovich Topunov1 1. A. N. Bach Institute of Biochemistry RAS, Moscow 119071, Russia 2. The Federal State Unitary Enterprise All-Russia Research Institute of Automatics, Moscow 127055, Russia Abstract: Rapid and reliable diagnostics and identification of pathogenic and symbiotic bacteria are at the top of the agenda. In the first case, they are important to control and prevent crop damages, and thus reduce economic losses. In the second, it’s necessary to design and monitor quality of biofertilizer to raise its effectiveness and crop capacity. Development of accurately, rapidly, technically and commercially accessible methods remains a critical problem for the bacteria with comprehensive phylogenetic structure. In this work, we investigated pathogenic Xanthomonas and Ralstonia and symbiotic Sinorhizobium. The aim of this investigation was to examine the applicability of the novel methods for phylogenetic study, identification and diagnostics of closely related species of these genera. The conventional phenotypic and genotypic (16S rRNA, gyrB) methods were applied as referents. Novel polymerase chain reaction (PCR)-based approaches, single-adapter amplified fragment length polymorphism (saAFLP) and comparative analyses of hin-region and Xcc0006-0007 sequences, were first employed for the investigations. Phenotypic tests, 16S rRNA and gyrB analysis distinguished bacteria at the genus level, but failed to identify them to the species robustly. The new methods identified bacteria at the inter-species level more precisely. This identification agreed with the accepted genera’s classifications. The only exceptions were X. fuscans & X. citri and X. perforance & X. euvesicatoria which clustered together. The further outcome of this study was achieved hin-region-based genus-specific PCR primers for the express-diagnostics of the genera. Summary, these new methods can be applied for genome-based phylogeny investigations and as convenient and accurate tools for identification and routine laboratory diagnostics of these comprehensive genera. Key words: Hin-region, saAFLP, Xanthomonas, Rhizobium, Sinorhizobium, Ralstonia. 1. Introduction beneficial associates and symbiotic microbes for biofertilizer design; (3) to provide surveillance and Plan-associated bacteria share a long history with control for both pathogenic and beneficial bacteria, plants and profound impact on the health and and thus prevent a bacterial diseases and increase a agricultural yield of crop [1]. There are two main crop yield. groups of bacteria based on their adverse/beneficial For a long time, bacterial classification has been effects on the host plants: pathogens and mutualists based largely on culture and biochemical tests, [2]. Investigation of the plant-bacterial interactions DNA-DNA hybridization, and 16S rRNA gene and microorganism’s taxonomic classification are the sequencing analysis [3]. Nowadays, classification, most important and developing directions of the identification and diagnostics integrate also new present biology [1]. The received knowledge can be techniques to determine both phenotypic and used in agricultural and biotechnological sectors: (1) genotypic characteristics [3-7]. The use of multilocus to diagnose most prevalent and most economically sequence typing (MLST), multilocus sequence important pathogens and prevent plant diseases; (2) to analysis (MLSA) and protein-typing are becoming identify and describe most common groups of very important for delineation of plant pathogenic and Corresponding author: Nataliya Vladimirovna Punina, symbiotic bacteria at species level and lower level [8]. Ph.D., research fields: microbiology and molecular biology. Over the last few years, these methods were improved E-mail: [email protected]. Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria 31 dramatically: increasingly mechanized to raise speed, methods were applied before [28]. There is still a need discrimination power and throughput, and to reduce for rapid, accurate, easy and low-cost approaches that cost of the laboratory analysis [9-16]. However, every can be applied to production for identification (control method has its limitations and selection of the and monitor) of the industrial strains. integrated approaches significantly depends on the In this work, the new DNA marker hin-region and target microorganism, time limits, assay accuracy and single-adapter amplified fragment length geographical scale [3, 17]. These methodical polymorphism (saAFLP) analysis were first used to disadvantages has become more complicated due to identify and describe the closely-related strains of the globalization, climate change, increased human genera Xanthomonas, Ralstonia and Sinorhizobium. mobility and bacterial evolution. Therefore, there is a Obtained results were compared with data derived by strong need to develop more rapid, accurate and the traditional pathogenicity and biochemical, universal techniques and methods to identify and comparative analysis of 16S rRNA, gyrB and detect economically important bacteria. Xcc0006-0007 nucleotide sequences. The potential of In this work, we studied two kinds of bacteria new methods for identification and diagnostics of characterized by the type of nutrition and agricultural strains was evaluated for these genera. importance: phytopathogenic genera Xanthomonas 2. Materials and Methods and Ralstonia and symbiotic Sinorhizobium. Xanthomonas and Ralstonia affect more than 400 2.1 Bacterial Strains species of agricultural crops, causing bacteriosis and The original and type strains from weeds and thus leading to major economic losses [18, 19]. Their agricultural crops were used in this study. One taxonomic structures are intricate and are being hundred and fourteen strains of Xanthomonas spp. constantly revised [20-22]. These genera had been were provided from All-Russian collection of subjected to numerous studies to clarify the taxonomy microorganisms, G. K. Skryabin Institute of and phylogeny, although all applied methods were Biochemistry and Physiology of Microorganisms partially sufficient for their identification and (VKM IBPM RAS; Pustchino, Russia), and Zabolotny diagnostics [21-27]. The disadvantage of the present Institute of Microbiology and Virology, National diagnostics of these genera is testing of the strain at Academy of Sciences of Ukraine (IMV; Kiev, the one taxonomic level and only a certain known Ukraine). Twelve Ralstonia sp. strains were isolated group of bacteria. Therefore, the development of new from wilted tomato and potato in Russia. The known rapid and efficient methods is still crucial for detection cultures of these genera obtained from the and identification of these phytopathogens. International Collection of Phytopathogenic Bacteria The genus Sinorhizobium (Ensifer) contains bacteria (ICPB; Davis, CA, USA), American Type Culture capable to fixate nitrogen in symbiosis with leguminous Collection (ATCC; Manassas, VA, USA) and plants including agricultural crops, e.g., soybeans [28]. National Collection of Plant-Pathogenic Bacteria This genus contains closely-related species, and some (NCPPB; York, England) were also included for of them are prospective for soybean biofertilizer reference. Two referential strains of nitrogen-fixing production [29]. Identification and characterization of Sinorhizobium meliloti B-117 and B-1009 were this genus are necessary to reveal and select most received from the collection of VKM IBPM RAS. efficient symbiotic bacteria, create most productive Nine strains of Sinorhizobium spp. were isolated from plant-symbiont combination and thus increase crop root nodules of field-grown soybeans in Russia in capacity of fields. Different phenotypic and genetic 2013. 32 Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria 2.2 DNA Isolation were amplified and sequenced using previously constructed primers UP1/UP2r and protocols [35]. The overall cellular DNA specimens were isolated from strains cultured on agarized yeast extract and 2.7 PCR Amplification and Sequencing of the tryptone (TY) medium (g/L): 5.0 g/L yeast extract, Xcc0006-0007 Region 10.0 g/L bacto-tryptone, 10.0 g/L NaCl and 20.0 g/L Nucleotide sequences of the Xcc0006-0007 region agar. DNA was isolated from cells on days 1-2 of of Xanthomonas spp. were amplified and sequenced culture by sorption onto magnetic particles (“Silex”, according previously published protocol [36]. Russia). 2.8 saAFLP Analysis 2.3 Phenotypic Characterization We modified amplified fragment length The morphological and biochemical characteristics polymorphism (AFLP) method developed before by of the pure bacterial cultures were determined based Vos et al. [37] and named new modification as on the general strategy of phenotypic differentiation saAFLP [38, 39]. The phylogenetic relationships described in Refs. [30-32]. between closely related strains Bacilllus cereus group 2.4 Pathogenicity Identification: Plant Nodulation Tests and Rhizobium leguminosarum had been successfully analyzed using this method [38, 39]. The procedure of All strains of genera Xanthomonas and Ralstonia saAFLP comprises three steps: (1) treatment of the were tested for their pathogenicity on wide host range using previously described
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